In animals, all of the enzymes in the pathway of de novo biosynthesis of fatty acids from malonyl-CoA are integrated into a homodimer of 540 kDa, the cytosolic fatty acid synthase complex (FAS). This pathway plays an essential role in embryogenesis and a key role in energy homeostasis and has been identified as a target for the development of both anti-obesity and anti-cancer therapeutic agents. The long-term objective of this program is to determine how the 14 functional domains of the dimer cooperate to synthesize palmitic acid. Compelling evidence has been obtained from several independent experimental approaches indicating that the classical view of FAS, as a pair of fully-extended, head-to-tail orientated subunits, is incorrect. This inference is supported by a 20 A-resolution structure derived by electron microscopy of individual FAS molecules labeled at the N-termini with gold particles. The proposed program is focused on correlation of topographical features of the electron microscope images with defined catalytic and structural elements of the complex and development of a detailed three-dimensional model based on characterization of 'nearest neighbor' relationships both within and between subunits. A group of investigators with unique expertise in FAS molecular biology and biochemistry, electron microscopy, bioorganic chemistry and mass spectrometry has been assembled to address these objectives. Several novel, complementary approaches will be exploited to introduce gold-cluster labels throughout the FAS, at the termini, in interdomain linkers and at active site residues, that will serve as site-specific markers in electron micrographs. The possibility that certain individual domains are dimeric will be tested using a combination of cross-linking, by both general and novel site-specific bifunctional reagents, and site-specific cleavage at engineered proteolytic sites. Insight into the spatial organization of the FAS will be sought by identifying 'nearest neighbor' relationships in the complex using cross-linking, proteolysis and mass spectrometry. A novel combination of 2H-labeled cross-linkers and heterodimeric FASs consisting of subunits separately labeled with 15N and 14N will be utilized to facilitate identification of derivatized peptides and allow distinction between inter- and intra-subunit cross-links. Results of the biochemical and mass spectrometry studies should provide a structural database that can be used to optimize interpretation of the electron micrographs and generate a detailed topographical map of the complex.